1. Field of the Invention
This invention relates to a polynucleotide or vector for expressing short interfering RNAs (siRNAs) to inhibit the expression of a target gene. The invention also relates to cells and non-human transgenic animals comprising the polynucleotide or vector and their various uses including in target drug validation and in human therapeutics.
The ability to inhibit or disrupt the function of a specific gene is highly desirable both from the point of view of studying gene function and also from a therapeutic perspective.
Many diseases arise from either the expression of a mutated gene or from abnormal, and in particular elevated or inappropriate, expression of a particular gene. Such mutations may be inherited, such as in the case of autosomal dominant disorders, or occur in the somatic or germ line tissues of an individual, such as in the case of cancer. The ability to modulate the expression of a mutated allele or of an inappropriately expressed wild type allele in various diseases or disorders may therefore be used to provide therapies to treat the disorders. In addition, in various infectious diseases, such as viral infection, the ability to inhibit the expression of viral genes in the host cell, or of a gene encoding a host cell protein involved in the life cycle of the virus, may also lead to possible treatments for infectious diseases.
The ability to inhibit gene expression has also been used to study gene function. Techniques such as classical mutagenesis have provided great insights into gene function, but such techniques are labour intensive, expensive and may take long periods of time. Such techniques simply may not be practical in higher organisms and require a means to identify the desired mutant. They also do not offer the possibility of mutating a specific gene of choice.
Although various methods for targeted gene disruption have been developed, where a gene of choice can be inhibited or disrupted, these also suffer from limitations. Techniques such as gene targeting are highly costly, expensive and time consuming often taking several years to obtain a homozygous mutant. Gene targeting also requires detailed knowledge of the structure of the gene to be disrupted.
As well as gene targeting antisense technology has also been developed to try and disrupt a specific gene. However, antisense RNA is unstable and it is often difficult to achieve high enough levels of antisense RNA in cells to achieve effective inhibition of a target gene.
Recently, it has been found in organisms such as C. elegans, Drosophilia melanogaster and plants that double stranded RNA molecules (dsRNA) are capable of inhibiting the expression of a target gene that they share sequence identity or homology to. The observed phenomena, sometimes referred to as post transcriptional gene silencing (PTGS), are thought to represent a possible cellular defence mechanism against viruses or transposons. Typically, in the studies carried out in these organisms the dsRNA has been introduced into cells by techniques such as microinjection or transfection and the inhibition of a target gene such as a reporter gene been measured.
The mechanism by which the dsRNA exerts its inhibitory effect on the target gene has begun to be elucidated. It is thought that the dsRNA is processed into duplexes of from 21 to 25 nucleotides in length. These short duplexes have been detected in plants where PTGS is occurring as well as in extracts of D. melanogaster schenider-2 (S2) cells transfected with a dsRNA molecule. It has been found that the processing reaction of a dsRNA can be carried out in vitro using extracts from these S2 cells. This provides an in vitro model system in which both the processing, targeting and transcript cleavage mechanisms involved in gene silencing can be studied. In the S2 lysate it was observed that the target mRNA was cleaved at 21 nucleotide intervals and that synthetic 21 and 22 RNA duplexes added to the lysate were able to guide efficient sequence specific mRNA degradation. Larger duplexes of 30 bp dsRNA were found to be active. The 21 nucleotide RNA products in the system were therefore named small interfering or silencing RNAs (siRNAs).
Factors from the target cell are also necessary for gene silencing. In D. melanogaster a ribonuclease III enzyme, dicer, is required for processing of the long dsRNAs into siRNA duplexes. It is thought that genes homologous to dicer exist in other organisms including mammals and humans as well as homologs or counterparts to the other host factors necessary. The initial steps in silencing involve the generation of a siRNA containing endonuclease complex. The endonuclease may be dicer or a gene homologous to dicer. The complex then specifically targets the mRNA transcript by a mechanism involving the exchange of one of the strands of the siRNA duplex with the region of sequence identity in the target transcript. Following this strand exchange, cleavage of the mRNA transcript occurs.
The cleavage of the target mRNA may occur at the ends of the duplexed region so, in effect, regenerating the siRNA endonuclease complex with one of the two strands of the regenerated siRNA coming from the original siRNA molecule and the other from the target transcript. Multiple cycles of transcript mRNA cleavage and hence siRNA regeneration may mean that each initial siRNA molecule can inactivate multiple copies of the target mRNA. Once the target mRNA transcript has been cleaved, the cleavage products not in the regenerated siRNA are rapidly degraded as they either lack the stabilising cap or pol(A)tail.
Although experiments investigating gene silencing in lower organisms have offered promising results it is thought that they may not be applicable to higher organisms such as mammals. It is thought that in higher organisms, such as mammals, cellular defence mechanisms operate which are triggered by dsRNA. It is believed that dsRNAs activate the interferon response which leads to a global shut-off in protein synthesis as well as non-specific mRNA degradation. This can lead to cell death and hence prevent selective gene inhibition. The presence of such defence mechanisms means that the applicability of gene silencing employing dsRNA in higher organisms has been questioned.
Experiments which have claimed to have demonstrated the efficacy of dsRNA in inhibiting the expression of a target gene in higher organisms have either been in non-mammalian systems, such as zebra fish or chicks, or alternatively in mammalian systems such as early embryos where the viral defence mechanisms are not thought to operate.
Preliminary experiments transfecting and/or microinjecting synthetic siRNAs, rather than longer dsRNA molecules which can be processed to give rise to a siRNA, have led to speculation that it might be possible to overcome the problems of the viral defence mechanisms in higher organisms. It may be that there is a threshold for the length of dsRNA necessary to activate the cell's defence mechanisms. The size of the synthetic siRNAs, and in particular the double stranded regions in them, introduced into the target cell may be small enough that they are below this threshold and hence do not activate the defence mechanisms.